The epsin N-terminal homology domain (ENTH) is a major player in clathrin-mediated endocytosis. To investigate the influence of initial membrane tension on ENTH binding and activity, we established a bilayer system based on adhered giant unilamellar vesicles (GUVs) to be able to control and adjust the membrane tension covering a broad regime. The shape of each individual adhered GUV as well as its adhesion area was monitored by spinning disc confocal laser microscopy. Control of in a range of 0.08-1.02 mN/m was achieved by altering the Mg 2؉ concentration in solution, which changes the surface adhesion energy per unit area of the GUVs. Specific binding of ENTH to phosphatidylinositol 4,5-bisphosphate leads to a substantial increase in adhesion area of the sessile GUV. At low tension (<0.1 mN/m) binding of ENTH can induce tubular structures, whereas at higher membrane tension the ENTH interaction deflates the sessile GUV and thereby increases the adhesion area. The increase in adhesion area is mainly attributed to a decrease in the area compressibility modulus K A . We propose that the insertion of the ENTH helix-0 into the membrane is largely responsible for the observed decrease in K A , which is supported by the observation that the mutant ENTH L6E shows a reduced increase in adhesion area. These results demonstrate that even in the absence of tubule formation, the area compressibility modulus and, as such, the bending rigidity of the membrane is considerably reduced upon ENTH binding. This renders membrane bending and tubule formation energetically less costly.Clathrin-mediated endocytosis is one of the key metabolic pathways for the uptake of macromolecules into eukaryotic cells (1-4). Driven by a chain of remodeling events and an elaborate set of proteins acting in an orchestrated manner, an almost flat patch of plasma membrane is transformed into a closed, cargo-containing vesicle. As plasma membrane shape transformation is associated with significant local bending of the membrane, the process is highly sensitive to lateral membrane tension. Plasma membrane tension originates from two primary sources; that is, hydrostatic pressure across the lipid bilayer and cytoskeleton-membrane adhesion (5). Depending on the cell type, plasma membrane tensions span a range of roughly 0.003-0.45 mN/m (5-8). Even though it had become clear already in the late 1990s that tension plays a role in exoand endocytosis (9, 10), only in recent years has significant evidence been accumulated that membrane tension is of utmost importance for processes that rely on membrane remodeling (11-14). Cells actively maintain and regulate their membrane tension and use it to control exo-and endocytosis (15). In K562 cells it has been reported that endocytosis is completely suppressed under hypoosmotic conditions (16). Generally, high lateral membrane tension suppresses membrane deformation as both stretching the lipid bilayer and opening bonds between the cytoskeleton and the plasma membrane requires a large amount of energy (17).One protein...

This is the first report showing that an epitope-specific ex vivo modulation of an allogeneic hematopoietic stem cell graft by the anti-human CD4 antibody MAX.16H5 IgG1 simultaneously facilitates the anti-tumor capacity of the graft (Graft-versus-leukemia effect, GvL) and the long-term suppression of the deleterious side effect Graft-versus-host-disease (GvHD). To distinguish and consolidate GvL from GvHD, the anti-human CD4 antibody MAX16.H5 IgG1 was tested in murine GvHD and tumor models. The survival rate was significantly increased in recipients receiving a MAX.16H5 IgG1 short-term (2 h) pre-incubated graft even when tumor cells were co-transplanted or when recipient mice were treated by MAX.16H5 IgG1 before transplantation. After engraftment, regulatory T-cells are generated only supporting the GvL effect. It was also possible to transfer the immune tolerance from GvHD-free recipient chimeras into third party recipient mice without the need of reapplication of MAX.16H5 IgG1 anti-human CD4 antibodies. These findings are also benefical for patients with leukemia when no matched related or unrelated donor is available and provides a safer allogeneic HSCT, which is more effective against leukemia. It also facilitates allogeneic (stem) cell transplantations for other indications (e.g., autoimmune-disorders).

Aims: We established a real-time PCR assay for the detection and strain identification of Candida species and demonstrated the ability to differentiate between Candida albicans the most common species, and also Candida parapsilosis, Candida glabrata, Candida tropicalis and Candida dubliniensis by LightCycler PCR and melting curve analysis. Methods and Results: The DNA isolation from cultures and serum was established using the QIAmp Tissue Kit. The sensitivity of the assay was >/= 2 genome equivalents/assay. It was possible to differentiate all investigated Candida species by melting curve analysis, and no cross-reaction to human DNA or Aspergillus species could be observed. Conclusions: The established real-time PCR assay is a useful tool for the rapid identification of Candida species and a base technology for more complex PCR assays. Significance and Impact of the Study: We carried out initial steps in validation of a PCR assay for the detection and differentiation of medically relevant Candida species. The PCR was improved by generating PCR standards, additional generation of melting curves for species identification and the possibility to investigate different specimens simultaneously

], the data showing the reconstitution of huCD4, HLA-DR, CD8, H2Kb, and muCD4 in the control animals that received 2 3 10 6 syngeneic bone marrow cells (Supporting Information Fig. S2-S4) were first published in Figure 5 of the reference 22:Fricke S, Fricke C, Oelkrug C, Hilger N, Sch€ onfelder U, Kamprad M, Lehmann J, Boltze J, Emmrich F, Sack U. Characterization of murine non-adherent bone marrow cells leading to recovery of endogenous hematopoiesis. Cell Mol Life Sci 2010;671231:4095-4106.The source article was not properly referenced in the legends of Figures S2, S3, and S4. This corrigendum is to correct this referencing error.In addition, the legend for Figure S3 was further modified by moving "(Day 0)" from the end of the second sentence explaining panel C to the beginning of the sentence, immediately following the words "After irradiation." The human CD4 molecule (only expressed on host T cells) decreases from initial 36.3% 6 3.66% to 8.20% 6 7.52% in co-transplanted animals and 23.23% 6 3.3% to 4.18% 6 0.43% in the bone marrow controls, respectively (Day 61). The controls in panels (A) and (B) were previously reported by us elsewhere (22). B: HLA-DR3 molecule (expressed on host APCs) decreases from initial 22.50% 6 6.73% to 0.18% 6 0.05% in co-transplanted animals and 20.98% 6 4.05% to 0.23% 6 0.05% in the bone marrow controls, respectively (Day 61). C: Flow cytometric dot plots of human CD4 (green) and HLA-DR3 (orange), and humanCD42/HLA-DR32 (red) on lymphocytes after syngeneic transplantation. Fig. S3: Transplantation of C57Bl/6 wild-type in C57Bl/6-TTG mice. The expression of murine CD31/CD81 on host cells after syngeneic transplantation. A: The expression of murine CD31/CD81 decreases after transplantation in the co-transplanted animals and in the bone marrow controls (13.35% 6 1.74% [Day 22] to 6.45% 6 3.33% [Day 19] vs.12.43% 6 2.46% [Day 22] to 4.48% 6 2.07% [Day 19]. The controls in panels (A) and (C) were previously reported by us elsewhere (22). After 61 days the initial CD8 levels were not reached. B: Flow cytometric dot plots of murine CD3 and murine CD8 on lymphocytes after syngeneic transplantation of BM or BM1Tregs. Murine CD31/CD81 are dark green events, murine CD31 are light green and pink events, and murine CD32 are black events. C: Murine MHC-H2Kb (from C57Bl/6) expression after syngeneic transplantation. The murine MHC-H2Kb was detectable at any time point during the experiment (over 95%). After irradiation (Day 0), the MHCH2Kb expression was 96.15% 6 3.5% in co-transplanted animals and 95.35% 6 2.96% in the bone marrow controls. D: Flow cytometric dot plots of murine MHC-H2Kb on lymphocytes after syngeneic transplantation of BM or BM1Tregs. All dots > 10 3 counts represent MHC-H2Kb1 cells, dots < 10 3 MHC-H2Kb2 cells.

Modern isothermal microcalorimeters (IMC) are able to detect the metabolic heat of bacteria with an accuracy sufficient to recognize even the smallest traces of bacterial contamination of water, food, and medical samples. The modern IMC techniques are often superior to conventional detection methods in terms of the detection time, reliability, labor, and technical effort. What is missing is a systematic analysis of the influence of the cultivation conditions on calorimetric detection. For the acceptance of IMC techniques, it is advantageous if already standardized cultivation techniques can be combined with calorimetry. Here we performed such a systematic analysis using Lactobacillus plantarum as a model bacterium. Independent of the cultivation techniques, IMC detections were much faster for high bacterial concentrations (>102 CFU⋅mL–1) than visual detections. At low bacterial concentrations (<102 CFU⋅mL–1), detection times were approximately the same. Our data demonstrate that all kinds of traditional cultivation techniques like growth on agar (GOA) or in agar (GIA), in liquid media (GL) or on agar after enrichment via membrane filtration (GF) can be combined with IMC. The order of the detection times was GF < GIA ≈ GL ≈ GOA. The observed linear relationship between the calorimetric detection times and the initial bacterial concentrations can be used to quantify the bacterial contamination. Further investigations regarding the correlation between the filling level (in mm) of the calorimetric vessel and the specific maximum heat flow (in μW⋅g–1) illustrated two completely different results for liquid and solid media. Due to the better availability of substrates and the homogeneous distribution of bacteria growing in a liquid medium, the volume-related maximum heat flow was independent on the filling level of the calorimetric vessels. However, in a solid medium, the volume-related maximum heat flow approached a threshold and achieved a maximum at low filling levels. This fundamentally different behavior can be explained by the spatial limitation of the growth of bacterial colonies and the reduced substrate supply due to diffusion.